The present invention relates to recombinant Yersinia and the use thereof for delivery of proteins into eukaryotic cells.
Bacteria of the genus Yersinia cause diseases in humans and rodents ranging from enteritis and lymphadenitis to plague. The genus Yersinia encompasses three species: Yersinia enterocolitica, which is the most prevalent Yersinia species in humans and causes a broad range of gastro-intestinal syndromes; Yersinia pseudotuberculosis, which causes adenitis and septicaemia; and Yersinia pestis, which is the causative agent of plague.
In spite of the differences in the infection routes, these three species of Yersinia share a common capacity to resist the non-specific immune response of the human or rodent host and to proliferate in the host lymphatic tissues. Anatomo-pathological examinations revealed that Yersinia are not detected inside the inflammatory or parenchymal cells of the infected animals (Simonet et al. (1990) Infect. Immun. 58: 841-845). Consistent with these in vivo observations, Yersinia are resistant to phagocytosis in vitro by macrophages and polymorphonuclear leukocytes. See review by Cornelis et al. (1997) Mol. Microbiol. 23(5): 861-867. Yersinia enterocolitica also has the capacity to enter certain cultured epithelial cells, a process generally referred to as invasion (Miller et al. (1988) Infect. Immun. 56: 1242-1248).
Genetic studies revealed that the virulence of Yersinia is determined by a 70 kb plasmid (pYV), which encodes and governs the production of a set of proteins called Yops (for Yersinia outer proteins). These Yops form an integrated anti-host system that allows the extracellular adhesion of Yersinia to the surface of host cells and the subsequent injection of a set of toxic effector proteins into the host cell""s cytosol. Recent studies further revealed that such an anti-host system, also called xe2x80x9cYersinia virulonxe2x80x9d, is composed of the following four elements: (i) a contact or type III secretion system called Ysc, which is devoted to the secretion of Yop proteins out of the bacterial cells; (ii) a set of xe2x80x9ctranslocatorsxe2x80x9d for translocating the effector proteins into the eukaryotic host cells, which consist of YopB, YopD and possible other proteins such as LcrV; (iii) a control element and recognition system (YopN and LcrG); and (iv) a set of xe2x80x9ceffector proteinsxe2x80x9d including YopE, YopH, YopO/YpkA, YopM and YopP/YopJ, which are injected (or translocated) into the eukaryotic host cells and disrupt the functions of such host cells. Transcription of these genes is controlled both by temperature and by contact with a eukaryotic cell. See review by Cornelis et al. (1997).
The effector proteins disrupt the function of host cells in a number of ways. The 23 kd YopE is a cytotoxin that disrupts the actin-microfilament structure of cultured Hela cells (Rosqvist et al. (1990) Mol. Microbiol. 4: 657-667; Rosqvist et al. (1991) Infect. Immun. 59: 4562-4569). The 51 kd YopH is a protein tyrosine phosphatase (PTPase) related to eukaryotic PTPases, which acts on tyrosine-phosphorylated proteins of infected macrophages (Hartland et al. (1994) Infect. Immun. 62: 4445-4453). Presumably as a result of this action, YopH inhibits bacterial uptake and oxidative burst by cultured macrophages (Rosquvist et al. (1988) Infect. Immun. 56: 2139-2143; Bliska et al. (1995) Infect. Immun. 63: 681-685). YopO (or YpkA) is an 81 kd serine/threonine kinase, which is targeted to the inner surface of the plasma membrane of the eukaryotic cell and might function to interfere with the signal transduction pathway of the eukaryotic cell (Hakansson et al. (1996) Mol. Microbiol. 20: 593-603). YopM is an acidic 41 kd protein having 12 leucine-rich repeats, which suggests that YopM might bind thrombin and interfere with platelet-mediated events of the inflammatory response (Leung et al. (1989) J. Bacterial. 171: 4623-4632). YopP is involved in the induction of apoptosis in macrophages (Mills et al. (1997) Proc. Acad. Natl. Sci. USA 94: 12638-12643).
The molecular structures of these effector proteins have been investigated to determine the elements in each effector protein that are required for their secretion and translocation. For this purpose, hybrid proteins have been engineered by fusing truncated Yop effector proteins of different length with certain reporter enzymes such as the calmodulin-activated adenylate cyclase domain (or Cya) of the Bordetella pertussis cyclolysin. Successful secretion and/or translocation events could be detected by assays based on the enzymatic activity of the reporter protein. Sory et al. disclose, by applying this approach, that YopE and YopH of Y. enterocolitica are modular proteins composed of three domains, i.e.,an N-terminal domain required for secretion, a translocation domain required for translocation into cells, and a C-terminal catalytic domain responsible for the toxic effector activity. Sory et al. (1995) Proc. Natl. Acad. Sci. USA 92: 11998-12002. The same domain organization has been demonstrated for YopM of Y. enterocolitca (Boland et al. (1996) EMBO J. 15: 5191-5201).
The present invention provides recombinant Yersinia for safe delivery of proteins into eukaryotic cells. Such Yersinia are deficient in the production of functional effector proteins, but are endowed with a functional secretion and translocation system. The present invention further provides expression vectors for use in combination with such mutant Yersinia for safe and efficient delivery of desired proteins into eukaryotic cells. This approach is useful not only for studying the function of a given protein, but also for designing therapeutic approaches. For example, a protein of a pathogenic origin, e.g., a tumor associated protein, a parasite antigen, or a viral antigen, can be delivered using the recombinant Yersinia of the present invention into antigen presenting cells for inducing an immune response specific for such a protein.
Most progressively growing neoplastic cells express potentially immunogenic tumor-associated antigens (TAAs), also called tumor rejection antigens (TRAs). TRAs, like other antigenic epitopes, are presented at the surface of tumor cells by MHC molecules and have been shown to induce a CTL response in vivo and in vitro. See, for example, van der Bruggen et al. (1991) Science 254: 1643-1647. However, such TRA-expressing tumor cells do not provoke reliable anti-tumor immune responses in vivo that are capable of controlling the growth of malignant cells. Boon et al. (1992) Cancer Surveys 13: 23-37; T. Boon (1993) Int. J. Cancer 54: 177-180; T. Boon (1992) Advances Cancer Res. 58: 177-209.
A number of genes have been identified that encode tumor rejection antigen precursors (or TRAPs), which are processed into TRAs in tumor cells. Such TRAP-encoding genes include members of the MAGE family, the BAGE family, the DAGE/Prame family, the GAGE family, the RAGE family, the SMAGE family, NAG, Tyrosinase, Melan-A/MART-1, gp100, MUC-1, TAG-72, CA125, mutated proto-oncogenes such as p21ras, mutated tumor suppressor genes such as p53, tumor associated viral antigens such as HPV16 E7. See, e.g., review by Van den Eynde and van der Bruggen (1997) in Curr. Opin. Immunol. 9:684-693, Sahin et al. (1997) in Curr. Opin. Immunol. 9:709-716, and Shawler et al. (1997) Advances in Pharmacology 40: 309-337 Academic Press, Inc.: San Diego, Calif. The identification of these genes has allowed recombinant production of TRAs or TRAPs which may be subsequently used as vaccines to treat various cancerous conditions.
The present invention contemplates the use of recombinant Yersinia for delivery of desired proteins into eukaryotic cells. Particularly, the recombinant Yersinia of the present invention are useful for delivery of proteins or derivatives thereof to antigen presenting cells. In accordance with the present invention, antigen presenting cells upon receiving the delivery, present antigenic epitopes which can be recognized by T cells. Thus, the recombinant Yersinia of the present invention can be employed in a number of immune diagnostic or therapeutic approaches.
The present invention is further elaborated upon the following disclosure.
The present invention relates to recombinant Yersinia and the use thereof for delivery of proteins into eukaryotic cells.
One embodiment of the present invention provides mutant Yersinia strains deficient in producing functional effector proteins. A preferred mutant Yersinia strain-of the present invention is a quintuple-mutant strain designated as yopEHMOP.
Another embodiment of the present invention provides expression vectors for delivery of heterologous proteins to eukaryotic cells. In accordance with the present invention, such an expression vector is characterized by (in the 5xe2x80x2 to 3xe2x80x2 direction) a promoter, a first nucleic acid sequence encoding a delivery signal, a second nucleic acid sequence fused thereto coding for a heterologous protein to be delivered.
According to this embodiment of the present invention, the promoter is preferably one from a Yersinia virulon gene; more preferably, an effector-encoding gene; even more preferably, a YopE gene. The delivery signal is a polypeptide sequence from an Yersinia effector, including YopE, YopH, YopO/YpkA, YopM, and YopP/YopJ. Such delivery signal can be recognized by the secretion and translocation system of Yersinia. The heterologous protein of the present invention includes naturally occurring proteins or parts thereof such as tumor-associated proteins or known antigens of pathogens. The heterologous protein of the present invention also includes artificially designed proteins such as in-frame fusion of proteins or parts of proteins.
Yet another embodiment of the present invention provides recombinant Yersinia, i.e., yersinia of the above-described mutant strains further transformed with the expression vector of the present invention. Such recombinant Yersinia is preferred for delivery of heterologous proteins into eukaryotic cells. A preferred eukaryotic cell is an antigen presenting cell capable of presenting immunogenic epitopes derived from the heterologous proteins being delivered.
In a further aspect of the present invention, such recombinant Yersinia are contemplated in immunogenic compositions and methods for inducing an immune response, either a cellular immune response or a humoral immune response, or a combination of both.
Further to this aspect of the invention, the recombinant Yersinia of the present invention can be employed in an in vitro regime for assessing the efficacy of a vaccination regimen. The recombinant Yersinia of the present invention can also be employed in an ex vivo regime to generate specific CTLs and to use such CTLs for treating various pathological conditions such as tumors or infections by pathogens. The recombinant Yersinia of the present invention can also be employed in an in vivo regime, i.e., as a recombinant vaccine, to treat subjects suffering pathological conditions such as tumors or infections by pathogens.